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Human SHMT Inhibitors Reveal Defective Glycine Import As a Targetable Metabolic Vulnerability of Diffuse Large B-Cell Lymphoma

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Human SHMT Inhibitors Reveal Defective Glycine Import As a Targetable Metabolic Vulnerability of Diffuse Large B-Cell Lymphoma Human SHMT inhibitors reveal defective glycine import as a targetable metabolic vulnerability of diffuse large B-cell lymphoma Gregory S. Duckera,b, Jonathan M. Ghergurovichb,c, Nello Mainolfid,VipinSurid, Stephanie K. Jeonga, Sophia Hsin-Jung Lic, Adam Friedmand, Mark G. Manfredid, Zemer Gitaic, Hahn Kima,e, and Joshua D. Rabinowitza,b,1 aDepartment of Chemistry, Princeton University, Princeton NJ 08544; bLewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton NJ 08544; cDepartment of Molecular Biology, Princeton University, Princeton NJ 08544; dRaze Therapeutics, Cambridge, MA 02139; and ePrinceton University Small Molecule Screening Center, Princeton University, Princeton, NJ 08544 Edited by Chi V. Dang, University of Pennsylvania School of Medicine, Philadelphia, PA, and accepted by Editorial Board Member Tak W. Mak September 6, 2017 (received for review April 20, 2017) The enzyme serine hydroxymethyltransferse (SHMT) converts serine One-carbon metabolism is targeted therapeutically by multiple into glycine and a tetrahydrofolate-bound one-carbon unit. Folate existing drugs, including the common clinical agents pemetrexed, one-carbon units support purine and thymidine synthesis, and thus 5-fluorouracil, and methotrexate (16). One mechanism of action cell growth. Mammals have both cytosolic SHMT1 and mitochon- common to several of these agents is inhibition of thymidylate syn- drial SHMT2, with the mitochondrial isozyme strongly up-regulated thase, which utilizes 5,10-methylene–THF. While new chemical tools in cancer. Here we show genetically that dual SHMT1/2 knockout have recently been disclosed that block de novo serine synthesis (17– blocks HCT-116 colon cancer tumor xenograft formation. Building 19), no existing chemotherapies specifically target the production of from a pyrazolopyran scaffold that inhibits plant SHMT, we identify 1C units from serine, the primary source of 1C units in tumors. ∼ small-molecule dual inhibitors of human SHMT1/2 (biochemical IC50 To block the production of 1C units from serine, simultaneous 10 nM). Metabolomics and isotope tracer studies demonstrate inhibition of both the cytosolic SHMT1 and mitochondrial SHMT2 effective cellular target engagement. A cancer cell-line screen is necessary. Here we genetically validate that dual SHMT1/2 genetic BIOCHEMISTRY revealed that B-cell lines are particularly sensitive to SHMT inhibi- knockout, in Ras-driven colon cancer cells, prevents xenograft for- tion. The one-carbon donor formate generally rescues cells from ’ mation. We present the development of a low nanomolar, stereo- SHMT inhibition, but paradoxically increases the inhibitor s cyto- specific small-molecule inhibitor of human SHMT1/2. Dual SHMT toxicity in diffuse large B-cell lymphoma (DLBCL). We show that inhibition blocks growth of many cell lines in a manner that is res- this effect is rooted in defective glycine uptake in DLBCL cell lines, cued by the soluble 1C donor formate. In diffuse large B-cell lym- rendering them uniquely dependent upon SHMT enzymatic activ- ity to meet glycine demand. Thus, defective glycine import is a phoma (DLBCL) cell lines, however, formate does not rescue cell targetable metabolic deficiency of DLBCL. growth but instead paradoxically enhances cancer cell death. We find that this unexpected outcome reflects a previously unappreciated SHMT | cancer metabolism | glycine | DLBCL | folate biochemical vulnerability of DLBCL: inability of these cells to ancer growth and proliferation are supported by metabolic Significance Cchanges, including enhanced glucose uptake, aerobic glycolysis (the Warburg effect), and folate-dependent one-carbon (1C) me- Enzymes of the folate cycle are among the most consistently tabolism (1, 2). The predominant source of 1C units in cancer cells overexpressed proteins in cancer. Whereas multiple clinical is the amino acid serine (3). The enzyme serine hydroxymethyl- agents inhibit thymidylate synthase, no current drugs target the transferase (SHMT) catalyzes the conversion of serine and tetra- incorporation of one-carbon into folates via serine hydroxy- hydrofolate (THF) into glycine and 5,10-methylene–THF. Increases methyltransferase (SHMT). Using genetics, we show that cancer in the synthesis and consumption of serine and glycine have been cells require SHMT to generate tumors. We then describe small- identified in transformed cells and cancers (4–6). Mitochondrial molecule SHMT inhibitors, and show that they block the growth SHMT (SHMT2) and the immediately downstream mitochon- of many human cancer cells, with B-cell lymphomas particularly drial enzyme 5,10-methylene-tetrahydrofolate dehydrogenase sensitive to SHMT inhibition. We find that this sensitivity arises (MTHFD2) are the most consistently overexpressed metabolic from the lymphomas’ inability to import the amino acid glycine, enzymes in cancer (7–9) (Fig. 1A). In most rapidly proliferating which is made as a byproduct of the SHMT reaction. Thus, B-cell cells, 1C units generated from serine catabolism in the mitochondria lymphomas have an intrinsic defect in amino acid import, which are exported to the cytosol as formate, which is then reassimilated causes a therapeutically targetable metabolic vulnerability. into folates to support nucleotide synthesis (10–12). While the mitochondrial pathway typically supplies all of the 1C Author contributions: G.S.D., N.M., V.S., S.H.-J.L., A.F., M.G.M., Z.G., H.K., and J.D.R. de- signed research; G.S.D., J.M.G., S.K.J., and S.H.-J.L. performed research; N.M. and H.K. units in proliferating cells in culture, it is not essential in nutrient contributed new reagents/analytic tools; G.S.D. J.M.G., S.H.-J.L., and V.S. analyzed data; replete conditions, as evidenced by the viability of SHMT2 and and G.S.D. and J.D.R. wrote the paper. MTHFD2 deletion cell lines (11, 13). In such deletion cells, cytosolic Conflict of interest statement: N.M., V.S., A.F., and M.G.M. are employees of Raze Ther- SHMT1 now metabolizes serine to produce 1C units required for apeutics. J.D.R. is a founder and member of the scientific advisory board of Raze Thera- purine and thymidine synthesis. However, the flux carried peutics. G.S.D., J.M.G., H.K., and J.D.R. are inventors on a Princeton University patent through this enzyme is insufficient to meet glycine demand, and covering serine hydroxymethyltransferse inhibitors and their use in cancer. mitochondrial folate-mutant cell lines are glycine auxotrophs This article is a PNAS Direct Submission. C.V.D. is a guest editor invited by the (14). Because glycine is abundant in serum, such auxotrophy has Editorial Board. not been considered physiologically relevant in mammals. How- Data deposition: The atomic coordinates and structure factors have been deposited in the ever, recent work has identified functional amino acid shortages Protein Data Bank, www.wwpdb.org (PDB ID code 5V7I). in human tumors, suggesting that transport from serum to tumor 1To whom correspondence should be addressed. Email: [email protected]. may be limiting in some contexts, resulting in dependence on This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. intracellular synthesis (15). 1073/pnas.1706617114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1706617114 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 from other cell lineages, or whether differential requirements A PHGDH mitochondria glucose 3-PG serine serine might exist for tumorigenesis versus maintenance. SHMT1 THF SHMT2 Small-Molecule Inhibitors of Human SHMT1/2. Compounds with the pyrazolopyran scaffold represented by compound 1 (Fig. 2A) were glycine glycine described as inhibitors of plant SHMT and showed efficacy as herbicides (20). Derivatives with a meta-thiophene substitution dTMP 5,10-meTHF 5,10-meTHF were recently published as inhibitors of Plasmodium SHMT (21). MTHFD1 MTHFD2 When these compounds were tested in human cell culture, potency was poor (22). We optimized compounds of this class for human purines 10-formylTHF 10-formylTHF SHMT1 and 2 (23). Compounds of this class were modestly more potent in vitro against SHMT1 than SHMT2. Changes that improve MTHFD1 MTHFD1L potency against both human isoforms include introduction of an iso- propyl group at the chiral four-carbon of the pyrano ring and adding formate steric bulk to the metasubstitutions on the phenyl ring (compound 2). BC ) ) 3 3 HCT-116 WT * Δshmt2 Δshmt2 Δshmt1/Δshmt2 * A * Plant SHMT inhibitor Optimized human SHMT inhibitors * Cl N OH Cl CF tumor volume (mm tumor volume (mm 3 days post injection days post injection CN CN CN Fig. 1. SHMT is required for tumor formation in vivo. (A) Serine synthesis and N N N catabolism occur in an intercompartmental cycle mediated by cytosolic and N N N O NH2 O NH2 O NH2 mitochondrial SHMT activity. Key enzymes mediating these transformations H H H are highlighted in capital letters. Arrows indicate the directionality of flux in (±)-1 (±)-2 (±)-3 HCT-116 cells, but most reactions are readily reversible. (B)Growthofsub- IC (nM) SHIN1 cutaneous tumors from HCT-116 WT and ΔSHMT2 cells implanted in opposite 50 flanks of nude mice (mean ± SEM, n = 10, *P < 0.05, paired t test). (C)Tumor SHMT1 593 13 5 growth of subcutaneous tumors from HCT-116 ΔSHMT2 and ΔSHMT1/2 cells SHMT2 1193 66 13 implanted in opposite flanks of nude mice (mean ± SEM, n = 10). B K409 Y105 take up glycine, which was previously viewed as a nonessential byproduct of the SHMT reaction. Results Y106 Requirement for SHMT Activity in HCT-116 Xenograft Formation. We generated clonal deletion cell lines of SHMT1, SHMT2, and SHMT1/ 2 from the human colorectal carcinoma cell line HCT-116. Paired GLY N410 Cas9 nickase (Cas9n)-containing constructs that encoded single-guide L166 RNA sequences targeting SHMT1 or -2 were transiently transfected 3.80Å into cells, and mutant colonies from single clones were picked as 2.99Å previously described (11). As previously reported, SHMT1 deletion H171 had no effect on cell growth either in cell culture or as subcutaneous SHMT2/ (2) SHMT1/ (5CHO-THF) xenografts in nude mice.
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